no / Bioactive lipid mediators derived from polyunsaturated fatty acids (PUFA) and exhibit a range of tissue and cell-specific activities in many physiological and pathological processes. Electrospray tandem mass spectrometry coupled to liquid chromatography (LC/ESI-MS/MS) is a sensitive, versatile analytical methodology for the qualitative and quantitative analysis of lipid mediators. Here we present an LC/ESI-MS/MS assay for the simultaneous analysis of twenty mono- and poly-hydroxy fatty acid derivatives of linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids. The assay was linear over the concentration range 1-100 pg/¿L, whilst the limits of detection and quantitation were 10-20 and 20-50 pg respectively. The recovery of the extraction methodology varied from 76-122% depending on the metabolite. This system is useful for profiling a range of biochemically-related potent mediators including the newly discovered resolvins and protectins, and their precursor hydroxy-eicosapentaenoic and hydroxy-docosahexaenoic acids, and, consequently, advance our understanding of the role of PUFA in health and disease. / Wellcome Trust, British Heart Foundation

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas. / Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas. / Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.